The Effects of Eugenol, Trans-Cinnamaldehyde, Citronellol, and Terpineol on Escherichia coli Biofilm Control as Assessed by Culture-Dependent and -Independent Methods.

Magdalena A Olszewska,Astrid Gędas,Manuel Simões

doi:10.3390/molecules25112641

Magdalena A Olszewska, Astrid Gędas + Show 1 more

Open Access

https://doi.org/10.3390/molecules25112641

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Abstract

Bacterial biofilms contribute to problems with preserving food hygiene, jeopardizing any conventional intervention method used by the food industry. Hence, the approach of using essential oil (EO) compounds effective in biofilm control has considerable merit and deserves in-depth research. In this study, the effect of selected EO compounds (eugenol, trans-cinnamaldehyde, citronellol, and terpineol) was assessed on Escherichia coli biofilm control by plate count, resazurin assay, and Syto® 9/PI (-/propidium iodide) staining coupled with flow cytometry (FCM) and confocal laser scanning microscopy (CLSM). The selected EO compounds effectively inhibited the growth of planktonic E. coli at low concentrations of 3–5 mM, revealing a high antimicrobial activity. EO compounds markedly interfered with biofilms too, with trans-cinnamaldehyde causing the most prominent effects. Its antibiofilm activity was manifested by a high reduction of cell metabolic activity (>60%) and almost complete reduction in biofilm cell culturability. In addition, almost 90% of the total cells had perturbed cell membranes. Trans-cinnamaldehyde further impacted the cell morphology resulting in the filamentation and, thus, in the creation of a mesh network of cells. Citronellol scored the second in terms of the severity of the observed effects. However, most of all, it strongly prevented native microcolony formation. Eugenol and terpineol also affected the formation of a typical biofilm structure; however, small cell aggregates were still repeatedly found. Overall, eugenol caused the mildest impairment of cell membranes where 50% of the total cells showed the Syto® 9+/PI– pattern coupled with healthy cells and another 48% with injured cells (the Syto® 9+/PI+). For terpineol, despite a similar percentage of healthy cells, another 45% was shared between moderately (Syto® 9+PI+) and heavily (Syto® 9–PI+) damaged cells. The results highlight the importance of a multi-method approach for an accurate assessment of EO compounds’ action against biofilms and may help develop better strategies for their effective use in the food industry.

Highlights

Bacterial biofilm formation is a serious and ongoing concern in the food industry, mostly due to the ability of biofilm inhabitants to withstand adverse conditions [1,2]
The antibacterial activity of essential oil (EO) compounds was determined in terms of MIC (Table 1)
In this study, selected EO compounds effectively inhibited the growth of planktonic E. coli at low concentrations of 3–5 mM, revealing a high antimicrobial activity and potential usability in the food industry

Summary

Introduction

Bacterial biofilm formation is a serious and ongoing concern in the food industry, mostly due to the ability of biofilm inhabitants to withstand adverse conditions [1,2]. Molecules 2020, 25, 2641 followed by microcolony formation and subsequent growth into mature biofilms with self-production of extracellular polymeric substances (EPS) [3]. They can be formed on all types of abiotic surfaces and on foods [4]. Bacterial adhesion on surfaces and subsequent biofilm formation usually leads to the enhanced antimicrobial tolerance of biofilm cells, while the risk of food contamination and/or recontamination is increased [5]. Even though the food safety risks related to biofilms have been extensively studied in the past two decades [6,7,8,9,10,11,12], biofilm formation in the food industry is still poorly controlled

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